Gastrointestinal (GI) peptides, including mammalian bombesin-like peptides, integrate secretory, contractile and proliferative functions in the GI tract. The broad, long term objective of this proposal remains to elucidate the signal transduction pathways that mediate GI peptide-mediated cell proliferation. GI peptides elicit their characteristic effects on cellular processes by binding to specific G protein-coupled receptors (GPCR) on the surface of their target cells. Protein kinase C (PKC) occupies a pivotal role in the signal transduction pathways that mediate numerous cellular responses elicited by GI peptides including cell proliferation. However, the events occurring downstream of specific isoforms of PKC remain elusive. The present challenge is to identify downstream targets for PKCs that transmit signals to the cell interior and participate in the regulation of processes that control cell function, including cell proliferation. We cloned a novel serine/threonine protein kinase, termed protein kinase D (PKD), with distinct structural and enzymological properties that has emerged as a downstream target of novel isoforms of PKC. During the past funding period, our studies established that the PKC/PKD pathway is operational in a variety of cell types stimulated with many different stimuli, generated experimental evidence supporting a model that envisages PKD activation as a dynamic multi-step process and indicated that PKD plays a role in the mediation of GPCR-induced cell proliferation. Based on these findings, our central hypothesis is that GI peptide receptors activate a novel PKC/PKD phosphorylation cascade that plays a critical role in the signal transduction of GI peptide-induced cellular proliferation. An additional hypothesis is that this novel PKC/PKD phosphorylation cascade is operational in gastrointestinal cells. We will examine these hypotheses pursuing the following specific aims: 1) Characterize the sequential multi-step mechanism of phosphorylation-dependent PKD activation in response to GPCR activation; 2) Identify the PKD domains necessary for potentiation of GPCR-induced cell proliferation; 3) Characterize the role of MAPK pathways in PKD potentiation of GPCR-induced cell proliferation; and 4) Characterize the regulation and function of PKD in intestinal epithelial cells.